Necrotizing enterocolitis (NEC) is a lethal gastrointestinal disease affecting premature infants. Although earlier studies have highlighted protective effects of milk-derived peptides against NEC, the role of the human β-casein-derived peptide BCCY-1 in intestinal barrier protection has never been investigated. Here, we showed that BCCY-1 alleviated the phenotype of NEC, reduced intestinal expression of Toll-like receptor 4 (TLR4) and interleukin-6, and improved the intestinal barrier integrity. NEC-associated multi-organ injury and impaired bone marrow hematopoiesis were also attenuated by BCCY-1. Metabolic screening revealed significant changes in intestinal metabolites in the NEC and NEC + BCCY-1 groups. Further analysis disclosed inhibition of 3-Nitrotyrosine formation due to the preservation of endothelial nitric oxide synthase (eNOS) activity, which was associated with the interactions between BCCY-1 and lipopolysaccharides, leading to disruption of TLR4 signaling. Our findings suggested that BCCY-1 improved intestinal barrier integrity through modulating the TLR4/eNOS/3-Nitrotyrosine axis, highlighting its potential role in the maintenance of intestinal health.
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